Limits...
Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in Escherichia coli.

Benhalevy D, Bochkareva ES, Biran I, Bibi E - PLoS ONE (2015)

Bottom Line: This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner.MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices.Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Are integral membrane protein-encoding mRNAs (MPRs) different from other mRNAs such as those encoding cytosolic mRNAs (CPRs)? This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner. MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices. To investigate this hypothesis, we designed DNA sequences encoding model untranslatable transcripts that mimic MPRs or CPRs. By utilizing in vitro-synthesized biotinylated RNAs mixed with Escherichia coli extracts, we identified a highly specific interaction that takes place between transcripts that mimic MPRs and the cold shock proteins CspE and CspC, which are normally expressed under physiological conditions. Co-purification studies with E. coli expressing 6His-tagged CspE or CspC confirmed that the specific interaction occurs in vivo not only with the model uracil-rich untranslatable transcripts but also with endogenous MPRs. Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

No MeSH data available.


Related in: MedlinePlus

6His-CspE/C pull-down experiments.E. coli cells co-expressing Ra, or Rb, or Rc, or Rd together with 6His-CspE or 6His-CspC were lysed, and extracts were incubated with Talon resin, for immobilizing 6His-CspE or 6His-CspC and their bound RNAs. (A) Samples from the various purification steps were analyzed by Westerm blotting with anti CspE antibodies. (B) Left panel, the eluates were treated with DNase or RNase and analyzed by Agarose gel. Right panel, the quality and size of the eluted RNA were analyzed by tapestation. (C) The total extract and the CspE/CspC bound RNAs were analyzed by qPCR with the corresponding primers to Ra, Rb, Rc, or Rd. Primers to rrl, rrs, and rnpB were utilized as controls. Values were calculated as 2extract Ct / 2pull-down Ct (see methods). Error bars indicate SEM (n = 3). (D) RNA was extracted from disrupted cells and the total steady state amount of R transcripts was measured by qPCR. Endogenous RNAs ssrA and rnpB were used as controls to assure unbiased results. Since both of the controls were similarly expressed in the various samples, we chose rnpB expression as a standard for calculating the relative quantity (RQ) of each R transcripts. The RQ of Ra is defined as = 1. (E) 6His-CspE (upper panel) or 6His-CspC (lower panel) were purified from wild type E. coli extracts with Talon resin. The total extracts and the eluates were analyzed by qPCR with primers to various MPRs and CPRs and analyzed as in (C). Error bars indicate SEM (n = 3). Right panels, an average ratio of [bound]/[total] is shown for each experiment.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520561&req=5

pone.0134413.g003: 6His-CspE/C pull-down experiments.E. coli cells co-expressing Ra, or Rb, or Rc, or Rd together with 6His-CspE or 6His-CspC were lysed, and extracts were incubated with Talon resin, for immobilizing 6His-CspE or 6His-CspC and their bound RNAs. (A) Samples from the various purification steps were analyzed by Westerm blotting with anti CspE antibodies. (B) Left panel, the eluates were treated with DNase or RNase and analyzed by Agarose gel. Right panel, the quality and size of the eluted RNA were analyzed by tapestation. (C) The total extract and the CspE/CspC bound RNAs were analyzed by qPCR with the corresponding primers to Ra, Rb, Rc, or Rd. Primers to rrl, rrs, and rnpB were utilized as controls. Values were calculated as 2extract Ct / 2pull-down Ct (see methods). Error bars indicate SEM (n = 3). (D) RNA was extracted from disrupted cells and the total steady state amount of R transcripts was measured by qPCR. Endogenous RNAs ssrA and rnpB were used as controls to assure unbiased results. Since both of the controls were similarly expressed in the various samples, we chose rnpB expression as a standard for calculating the relative quantity (RQ) of each R transcripts. The RQ of Ra is defined as = 1. (E) 6His-CspE (upper panel) or 6His-CspC (lower panel) were purified from wild type E. coli extracts with Talon resin. The total extracts and the eluates were analyzed by qPCR with primers to various MPRs and CPRs and analyzed as in (C). Error bars indicate SEM (n = 3). Right panels, an average ratio of [bound]/[total] is shown for each experiment.

Mentions: The above studies were performed with biotinylated RNA molecules prepared in vitro. To determine whether the CspE/C-U-rich RNA interaction also occurs in vivo with non-biotinylated RNAs, cells were co-transformed with a plasmid encoding Ra, Rb, Rc, or Rd together with a compatible plasmid encoding 6His-CspE or 6His-CspC. Cell extracts were incubated with Talon beads and the eluted, purified 6His-CspE-RNA or 6His-CspC-RNA complexes were analyzed by Western blotting with anti-CspE antibodies (Fig 3A), total RNA characterization (Fig 3B) and qPCR (Fig 3C and 3E and S2 Fig). Fig 3A shows Western blot analysis of samples from the purification steps, using anti-CspE antibodies. This analysis revealed that the endogenous CspE and CspC proteins (lower bands in Fig 3A) are relatively highly expressed even in cells harboring plasmid-encoded 6His-tagged CSPs. In the case of the 6His-CspE, we analyzed the presence of nucleic acids in the eluate, and the results show clearly that it contains only RNA (Fig 3B, left panel), which was characterized by the tapestation for both eluates (Fig 3B, right panels). The Tapestation analysis also indicates that the 6His-CspC and 6His CspE bound material is highly enriched with RNAs. The input and eluted RNAs were then analyzed by qPCR using R-transcripts or control primers (Fig 3C), and the results show unequivocally that the specific interaction between CSPs and U-rich RNAs (Ra and Rc) is maintained also in vivo and is biotinylation-independent. During the subcellular distribution experiments (Fig 1C and 1D), we observed that the expression of U-rich RNAs was specifically low and could have major effects on our pull-down experiments. To test this quantitatively, we measured the amount of the model transcripts at steady state. Fig 3D shows that indeed, the steady-state levels of the U-rich transcripts Ra and Rc were very low compared with those of Rb and Rd. This finding markedly strengthens our conclusion that CspE and CspC interact specifically with the U-rich transcripts, despite their low expression.


Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in Escherichia coli.

Benhalevy D, Bochkareva ES, Biran I, Bibi E - PLoS ONE (2015)

6His-CspE/C pull-down experiments.E. coli cells co-expressing Ra, or Rb, or Rc, or Rd together with 6His-CspE or 6His-CspC were lysed, and extracts were incubated with Talon resin, for immobilizing 6His-CspE or 6His-CspC and their bound RNAs. (A) Samples from the various purification steps were analyzed by Westerm blotting with anti CspE antibodies. (B) Left panel, the eluates were treated with DNase or RNase and analyzed by Agarose gel. Right panel, the quality and size of the eluted RNA were analyzed by tapestation. (C) The total extract and the CspE/CspC bound RNAs were analyzed by qPCR with the corresponding primers to Ra, Rb, Rc, or Rd. Primers to rrl, rrs, and rnpB were utilized as controls. Values were calculated as 2extract Ct / 2pull-down Ct (see methods). Error bars indicate SEM (n = 3). (D) RNA was extracted from disrupted cells and the total steady state amount of R transcripts was measured by qPCR. Endogenous RNAs ssrA and rnpB were used as controls to assure unbiased results. Since both of the controls were similarly expressed in the various samples, we chose rnpB expression as a standard for calculating the relative quantity (RQ) of each R transcripts. The RQ of Ra is defined as = 1. (E) 6His-CspE (upper panel) or 6His-CspC (lower panel) were purified from wild type E. coli extracts with Talon resin. The total extracts and the eluates were analyzed by qPCR with primers to various MPRs and CPRs and analyzed as in (C). Error bars indicate SEM (n = 3). Right panels, an average ratio of [bound]/[total] is shown for each experiment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520561&req=5

pone.0134413.g003: 6His-CspE/C pull-down experiments.E. coli cells co-expressing Ra, or Rb, or Rc, or Rd together with 6His-CspE or 6His-CspC were lysed, and extracts were incubated with Talon resin, for immobilizing 6His-CspE or 6His-CspC and their bound RNAs. (A) Samples from the various purification steps were analyzed by Westerm blotting with anti CspE antibodies. (B) Left panel, the eluates were treated with DNase or RNase and analyzed by Agarose gel. Right panel, the quality and size of the eluted RNA were analyzed by tapestation. (C) The total extract and the CspE/CspC bound RNAs were analyzed by qPCR with the corresponding primers to Ra, Rb, Rc, or Rd. Primers to rrl, rrs, and rnpB were utilized as controls. Values were calculated as 2extract Ct / 2pull-down Ct (see methods). Error bars indicate SEM (n = 3). (D) RNA was extracted from disrupted cells and the total steady state amount of R transcripts was measured by qPCR. Endogenous RNAs ssrA and rnpB were used as controls to assure unbiased results. Since both of the controls were similarly expressed in the various samples, we chose rnpB expression as a standard for calculating the relative quantity (RQ) of each R transcripts. The RQ of Ra is defined as = 1. (E) 6His-CspE (upper panel) or 6His-CspC (lower panel) were purified from wild type E. coli extracts with Talon resin. The total extracts and the eluates were analyzed by qPCR with primers to various MPRs and CPRs and analyzed as in (C). Error bars indicate SEM (n = 3). Right panels, an average ratio of [bound]/[total] is shown for each experiment.
Mentions: The above studies were performed with biotinylated RNA molecules prepared in vitro. To determine whether the CspE/C-U-rich RNA interaction also occurs in vivo with non-biotinylated RNAs, cells were co-transformed with a plasmid encoding Ra, Rb, Rc, or Rd together with a compatible plasmid encoding 6His-CspE or 6His-CspC. Cell extracts were incubated with Talon beads and the eluted, purified 6His-CspE-RNA or 6His-CspC-RNA complexes were analyzed by Western blotting with anti-CspE antibodies (Fig 3A), total RNA characterization (Fig 3B) and qPCR (Fig 3C and 3E and S2 Fig). Fig 3A shows Western blot analysis of samples from the purification steps, using anti-CspE antibodies. This analysis revealed that the endogenous CspE and CspC proteins (lower bands in Fig 3A) are relatively highly expressed even in cells harboring plasmid-encoded 6His-tagged CSPs. In the case of the 6His-CspE, we analyzed the presence of nucleic acids in the eluate, and the results show clearly that it contains only RNA (Fig 3B, left panel), which was characterized by the tapestation for both eluates (Fig 3B, right panels). The Tapestation analysis also indicates that the 6His-CspC and 6His CspE bound material is highly enriched with RNAs. The input and eluted RNAs were then analyzed by qPCR using R-transcripts or control primers (Fig 3C), and the results show unequivocally that the specific interaction between CSPs and U-rich RNAs (Ra and Rc) is maintained also in vivo and is biotinylation-independent. During the subcellular distribution experiments (Fig 1C and 1D), we observed that the expression of U-rich RNAs was specifically low and could have major effects on our pull-down experiments. To test this quantitatively, we measured the amount of the model transcripts at steady state. Fig 3D shows that indeed, the steady-state levels of the U-rich transcripts Ra and Rc were very low compared with those of Rb and Rd. This finding markedly strengthens our conclusion that CspE and CspC interact specifically with the U-rich transcripts, despite their low expression.

Bottom Line: This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner.MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices.Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Are integral membrane protein-encoding mRNAs (MPRs) different from other mRNAs such as those encoding cytosolic mRNAs (CPRs)? This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner. MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices. To investigate this hypothesis, we designed DNA sequences encoding model untranslatable transcripts that mimic MPRs or CPRs. By utilizing in vitro-synthesized biotinylated RNAs mixed with Escherichia coli extracts, we identified a highly specific interaction that takes place between transcripts that mimic MPRs and the cold shock proteins CspE and CspC, which are normally expressed under physiological conditions. Co-purification studies with E. coli expressing 6His-tagged CspE or CspC confirmed that the specific interaction occurs in vivo not only with the model uracil-rich untranslatable transcripts but also with endogenous MPRs. Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

No MeSH data available.


Related in: MedlinePlus